Differential impact of asparaginase discontinuation on outcomes of children with T‐cell acute lymphoblastic leukemia and T‐cell lymphoblastic lymphoma

Abstract Background Asparaginase is essential for treating T‐cell acute lymphoblastic leukemia (T‐ALL). Despite the ongoing debate on whether T‐ALL and T‐cell lymphoblastic lymphoma (T‐LBL) are the same disease entity or two distinct diseases, patients with T‐LBL often receive the same or similar treatment protocols as those with T‐ALL. Methods The outcomes of patients with or without L‐asparaginase discontinuation were retrospectively analyzed among four national protocols: Japan Association of Childhood Leukemia Study (JACLS) ALL‐02 and ALL‐97 for T‐ALL and Japanese Pediatric Leukemia/Lymphoma Study Group ALB‐NHL03 and JACLS NHL‐98 for T‐LBL. The hazard ratio (HR) was calculated with the Cox regression model by considering L‐asparaginase discontinuation as a time‐dependent variable. Results In total, 199 patients with T‐ALL, and 133 patients with T‐LBL were included. L‐asparaginase discontinuation compromised event‐free survival (EFS) of T‐ALL patients (ALL‐02: HR 3.32, 95% confidence interval [CI] 1.40–7.90; ALL‐97: HR 3.39, 95%CI 1.19–9.67). Conversely, EFS compromise was not detected among T‐LBL patients (ALB‐NHL03: HR 1.39, 95%CI 0.41–4.68; NHL‐98: HR 0.92, 95%CI 0.11–7.60). Conclusion The effects of L‐asparaginase discontinuation differed between T‐ALL and T‐LBL. We assume that the differential impact results from (1) the inherent differential response to L‐asparaginase between them and/or (2) a less stringent assessment of early treatment response in T‐LBL than in T‐ALL. Given the poor salvage rate of refractory or relapsed T‐ALL and T‐LBL, optimization of the frontline therapy is critical, and the current study provides a new suggestion for further treatment modifications. However, larger studies in contemporary intensified treatment protocols are required.


| INTRODUCTION
Asparaginase plays an important role in the chemotherapeutic regimens to treat acute lymphoblastic leukemia (ALL).However, asparaginase is frequently discontinued due to its unique adverse effects, such as clinical allergy, subclinical hypersensitivity, acute pancreatitis, and thrombosis. 1,2Asparaginase has been thought to be even more effective and important for T-cell ALL than for B-cell ALL, 3,4 and we have previously identified that L-asparaginase discontinuation was associated with compromised outcomes among children with T-ALL. 5owever, our previous study analyzed only one protocol and included 79 patients with T-ALL who achieved remission after induction therapy; thus, external validation would be warranted.
The discussion about whether T-ALL and T-cell lymphoblastic lymphoma (T-LBL) are a spectrum of the same disease or two distinct disease entities is ongoing.Remarkable similarities have been suggested regarding clinical or cytogenetic characteristics, 6 and the World Health Organization and the International Lymphoma Study Group classify these two diseases into one entity, that is, T-lymphoblastic leukemia/lymphoma. 7,80][11][12][13][14] On the contrary, several recent clinical and biological data raised the possibility that T-ALL and T-LBL are different disease entities. 15They differ in terms of immunophenotypes, 16 gene expression profiles, 17,18 and DNA methylation characteristics. 19Furthermore, patients with cancer predisposition syndromes predispose to each of T-ALL or T-LBL specifically.Ataxia telangiectasia predisposes to T-ALL and mature B-cell non-Hodgkin lymphomas but rarely to T-LBL. 20,21Conversely, Nijmegen breakage syndrome predisposes to lymphomas including T-LBL but less frequently to T-ALL. 22In this context, the effect of discontinuation of asparaginase may differ between these two diseases, and the impact of asparaginase discontinuation in children with T-LBL should be investigated as well, and should be compared to those with T-ALL.
Here, we analyzed the characteristics and outcomes of patients with or without L-asparaginase discontinuation among four national protocols for T-ALL and T-LBL: Japan Association of Childhood Leukemia Study (JACLS) ALL-02 and ALL-97 for T-ALL and Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) ALB-NHL03 and JACLS NHL-98 for T-LBL.

| Patients
The current study included clinical data of patients with T-ALL or advanced stage (III or IV) T-LBL extracted from four treatment protocols: JACLS ALL-02, JCACLS ALL-97, JACLS NHL-98, and JPLSG ALB-NHL03 (UMIN000002212, http:// www.umin.ac.jp/ ctr/ index -j.4][25]   .Accordingly, we included those with T-ALL and T-LBL who were treated in similar years.Each patient's guardian provided informed consent per the Declaration of Helsinki.The Ethical Committees of all participating institutions approved the trials.][25] Previously, we investigated the effects of L-asparaginase discontinuation on patients treated with JACLS ALL-02 protocols 5 ; in that study, we analyzed patients with T-ALL who showed <25% blast at day15 bone marrow, achieved remission after induction, and thus received treatment based on the T-02 protocol.On the contrary, in the current study, we analyzed all patients with T-ALL.

| Treatment protocols
The details and results of these four protocols have been previously published, 5,[23][24][25] and the details are also summarized in Tables S1-S4 and Figure S1.L-asparaginase schedules of each protocol are described as follows.Lasparaginase was the only preparation of asparaginase approved for use in Japan during the study periods of these four protocols, and Erwinia asparaginase (Erwinase) could not replace the native L-asparaginase in case of discontinuation.In all four protocols, L-asparaginase was discontinued in case of systemic allergy reaction, acute pancreatitis, or asparaginase-related complications for which the treating physician decided to discontinue asparaginase.These reasons and the timing of asparaginase discontinuation were centrally reported.Subclinical hypersensitivity was not measured in these clinical studies.Additional drugs were administered only in the ALL-02 protocol after Lasparaginase discontinuation.No treatment modification was applied besides L-asparaginase discontinuation in the other protocols.The total doses of L-asparaginase of these protocols are summarized in Table 1.
JACLS ALL-02: Among patients with T-ALL, those who showed blast <25% at Day 15 bone marrow and achieved remission after induction received treatment based on T-02 protocols (n = 84), while those who failed to achieve above criteria were allocated to F protocol (n = 23).The induction phase comprised six doses of L-asparaginase (6000 U/ m 2 per dose) administered for 12 days.In T-02 protocol, patients received L-asparaginase therapy (6000 U/m 2 per dose) for 5 consecutive days during the sanctuary therapy.They also received two courses of six doses of intermittent Lasparaginase (10,000 U/m 2 ) and two courses of three doses of L-asparaginase (10,000 U/m 2 ) for 14 days during the maintenance therapy.If L-asparaginase was discontinued, patients received one course of intensified therapy, including highdose cytarabine, etoposide, and PSL, immediately before the initiation of maintenance therapy.In F protocol, after induction phase, patients received two courses of consolidation therapy, each containing five doses of L-asparaginase (6000 U/m 2 ).JACLS ALL-97 and JACLS NHL-98: Both protocols applied the same schedules of L-asparaginase therapy as described previously. 23Among patients with T-ALL, those who showed blast <5% at Day 15 bone marrow and achieved remission after induction received treatment based on T-97 protocols (n = 61), while those who failed to achieve above criteria were allocated to F protocol (n = 11).In summary, in ALL-97 and NHL-98 protocols, the induction phase comprised six doses of L-asparaginase (10,000 U/ m 2 per dose) administered for 12 days, followed by consolidation therapies A and B, which both contained five doses of L-asparaginase (10,000 U/m 2 per dose) administered for 5 consecutive days.Patients received consolidation therapies A and B twice.During the maintenance therapy, patients received block B thrice, block C twice, and block D thrice.Block C comprised five doses of L-asparaginase (10,000 U/ m 2 per dose) administered for 5 consecutive days.Blocks B and D contained three L-asparaginase doses (10,000 U/m 2 per dose) administered for 14 days.JPLSG ALB-NHL03: The induction phase comprised nine doses of L-asparaginase (6000 U/m 2 per dose) administered for 19 days.During the reinduction therapy, six doses of L-asparaginase (6000 U/m 2 per dose) were administered for 12 days.A total of fourteen doses of L-asparaginase (6000 U/m 2 per dose) were administered during the early maintenance therapy, and 10 doses (6000 U/m 2 per dose) were administered during the late maintenance therapy.

| Statistical analysis
Event-free survival (EFS) and overall survival (OS) were defined as the time from diagnosis to the date of an event, where events were defined as death from any cause for OS and as first relapse, death, induction failure, or secondary neoplasm for EFS.Induction failure was defined as patients who did not achieve <5% blasts in the bone marrow for those with T-ALL and those who did not achieve complete remission (CR), unconfirmed remission, or partial response for T-LBL.Induction failure was defined as an event at the predefined evaluation dates after the induction therapy: day 33 for JACLS ALL-02 and JPLSG ALB-NHL03 and day 35 for JACLS ALL-97 and NHL-98.Competing events were defined as death without relapse for relapse and relapse for nonrelapse mortality.Hazard ratio (HR) for EFS was calculated using extended Cox regression model, and HR for incidence of relapse was calculated using Fine and Gray competing risk regression model by considering asparaginase discontinuation as a time-dependent variable. 26Statistical analyses were conducted using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), 27 a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria).p < 0.05 was considered statistically significant for all analyses.

| Patient characteristics
Patient characteristics across four treatment protocols are described in The reasons for L-asparaginase discontinuation are shown in Table 3, with acute pancreatitis being the most frequent as observed in 16/179 (6.7%) patients with T-ALL and 16/133 (12.0%) patients with T-LBL.Discontinuation due to allergy or thrombosis was rare, as only seen in <3% of patients in each protocol.The majority of patients discontinued L-asparaginase before the start of the maintenance therapy phase, except for those in the NHL-98 protocol (Table S5).Abbreviations: ALL, acute lymphoblastic leukemia; LBL, lymphoblastic lymphoma.

| Survival analysis
The The HR was calculated by considering L-asparaginase discontinuation as a time-dependent variable.As shown in Figure 1, EFS was compromised when L-asparaginase was discontinued in the JACLS ALL-02 (HR 3.32, 95% CI 1.40-7.90)and JACLS ALL-97 (HR 3.39, 95% CI 1.19-9.67)protocols.Conversely, EFS compromise was not detected in the JPLSG ALB-NHL03 (HR 1.39, 95% CI 0.41-4.68)and JACLS NHL-98 (HR 0.92, 95% CI 0.11-7.60)protocols.These results were maintained in the analysis for cumulative incidence of relapse (CIR).L-asparaginase discontinuation was associated with worse CIR in T-ALL patients, but not as clear in T-LBL patients (Figure 2).The overall trend was less evident when the HR for OS was calculated (Figure S2).

| DISCUSSION
Although the patient outcomes were significantly compromised when L-asparaginase was discontinued in the two independent protocols for T-ALL, the outcome was not altered when L-asparaginase was discontinued in the two independent protocols for T-LBL.14]28 Given the poor F I G U R E 1 Hazard ratio of L-asparaginase discontinuation on event-free survival according to the treatment protocols.CI, confidence interval; L-Asp, L-Asparaginase.

F I G U R E 2
Hazard ratio of L-asparaginase discontinuation on cumulative incidence of relapse according to the treatment protocols.CI, confidence interval; L-Asp, L-Asparaginase.salvage rate of refractory or relapsed T-ALL and T-LBL, 11 optimization of the frontline therapy is critical, and the current study provided a new suggestion for further treatment modifications.
Our previous study using the cohort of JACLS ALL-02 study showed that those with T-ALL suffered compromised outcomes when L-asparaginase was discontinued. 5n the current study, we expanded the inclusion criteria in the JACLS ALL-02 protocol cohort and also referred to the independent ALL-97 cohort.As shown in Figure 1, the compromised outcomes of those with T-ALL were observed in both JACLS ALL-02 and ALL-97 cohorts.The background characteristics of the included patients in the two studies generally aligned with previous studies.The frequencies of central nervous system involvement at diagnosis were also similar to those of a previous study. 6oreover, the total doses of L-asparaginase in JACLS ALL-97 were larger than those of ALL-02 (Table 1), which we believed further supported the detrimental effect of Lasparaginase discontinuation.As such, we assumed that we successfully confirmed the result of our previous study, where those with T-ALL suffered compromised outcomes when L-asparaginase was discontinued. 5he most striking result in the current study was the different effects of L-asparaginase discontinuation between patients with T-ALL and T-LBL.In contrast to T-ALL, the effect of L-asparaginase discontinuation was much less clear in patients with T-LBL in the current study.This was in line with the recent large clinical studies of the Children's Oncology Group (COG), highlighting differential responses to chemotherapy between T-ALL and T-LBL.3][14] Interestingly, nelarabine improved the survival of patients with T-ALL but not of those with T-LBL in the AALL0434 trial.Meanwhile, bortezomib improved the survival of patients with T-LBL but not of those with T-ALL in the AALL1231 trial.These results suggested that T-ALL and T-LBL have different responses to specific treatment drugs, and we suspected that the response to Lasparaginase might also be different between T-ALL and T-LBL.Furthermore, the study suggests the potential for varying effects of replacement therapies following asparaginase discontinuation between T-ALL and T-LBL, as children with T-ALL would benefit more from such therapies.
Another possible explanation for this differential effect of L-asparaginase discontinuation might be the difference in assessment measures of treatment responses between these two diseases.Among patients with T-ALL, the response is measured by prednisone response, bone marrow morphology, or, more recently, polymerase chain reaction-based, or flow-cytometry-based minimal residual disease detection.However, the response is usually measured by the tumor size changes evaluated with computed tomography in T-LBL, and the stage at diagnosis is the only parameter for risk group stratification, classifying patients into limited (stage I and II) versus advanced (stage III and IV) stages.Accordingly, the evaluation of treatment response in T-LBL might not be as stringent as in T-ALL, and patients with T-LBL who had suboptimal treatment response might continue to be included in the first-line protocol without any intensification and would experience relapse subsequently, with or without L-asparaginase discontinuation.Meanwhile, a recent study investigating the utility of fluoro-deoxy-glucose positron emission tomography (PET-CT) showed a significant positive correlation between minimal residual disease and patient outcomes among those with ALL and LBL. 29dentifying patients with early suboptimal treatment responses using a more refined method such as PET-CT 30 might be critically needed for those with T-LBL, but this should be evaluated in a prospective clinical trial.
As shown in Table 3, the incidence of acute pancreatitis differed based on treatment protocols.Asparaginaseassociated pancreatitis occurs in 2%-18% of patients treated for ALL, [31][32][33][34][35][36] and a previous study by Liu et al. identified older age and higher cumulative doses of asparaginase as risk factors of asparaginase-associated acute pancreatitis. 33As shown in Tables 1 and 3, our study also demonstrated that the frequency of acute pancreatitis in protocols with higher doses of L-asparaginase (JACLS ALL-97; 13.9% and JACLS NHL-98; 27.6%) seemed higher than that of protocols with lower doses (JACLS ALL-02; 1.9% and JPLSG ALB-NHL03; 7.7%), regardless of disease types.The frequency of L-asparaginase discontinuation due to allergy was low among those receiving ALL-02, while the frequency among those receiving ALL-97 was similar to those of other countries. 37We attributed this difference in treatment schedules, and this was already discussed in our previous report. 5his study has several limitations.First, four different protocols were included.These differences make it difficult to directly compare the effects of L-asparaginase discontinuation between T-ALL and T-LBL, as the differential effect of discontinuing asparaginase could depend more on protocol differences rather than on biological differences between T-ALL and T-LBL.In fact, in the NHL-98 protocol, L-asparaginase was discontinued more often during maintenance therapy than during the premaintenance phase (Table S5).This could pose a challenge in identifying the detrimental effects of L-asparaginase discontinuation.Moreover, due to data limitations, exact doses before L-asparaginase discontinuation could not be analyzed, hindering comparison among the four studies.
However, it is worth mentioning that the detrimental effect of L-asparaginase discontinuation was not observed among those who received the ALB-NHL03 protocol, in which most patients discontinued L-asparaginase before the start of the maintenance therapy.We are considering applying the same or similar treatment for T-ALL and T-LBL in Japan and expect that this strategy will allow us to directly compare the effects of asparaginase discontinuation.Second, as the survival outcomes of our protocols for T-ALL were suboptimal compared to the contemporary protocols from other cooperating groups, [12][13][14]38,39 the effect of asparaginase discontinuation might differ in the context of contemporary treatment protocols. Third Lasparaginase was the only preparation approved for use in Japan during the study period of these protocols, and the results might be different when using PEG-asparaginase, which is much more widely used nowadays.Finally, we think the statistical results in this manuscript should be evaluated with caution.Initially, we attempted to analyze the effect of discontinuation using a landmark-based method, as we did in the previous study.However, this did not seem feasible because many events occurred within the treatment period for those with T-LBL in this study, making it difficult to set a suitable landmark for them.As our conclusion was based solely on one method, 26 we suggest that the results should be further evaluated in external cohorts.
In conclusion, while the patient outcomes were significantly compromised when L-asparaginase was discontinued in the two independent protocols for T-ALL, the outcome did not differ when L-asparaginase was discontinued in the two independent protocols for T-LBL.We assumed that this differential impact of L-asparaginase discontinuation resulted from (1) the inherent differential response to L-asparaginase between T-ALL and T-LBL and/or (2) less strict assessment of early treatment response among T-LBL than in T-ALL.Given a poor salvage rate of refractory or relapsed T-ALL and T-LBL, optimization of the frontline therapy is critical, and the current study provided a new suggestion for further treatment modifications.Further studies with larger patient numbers in contemporary intensified treatment protocols are required to confirm this differential impact between T-ALL and T-LBL.
inherent differential response to L-asparaginase between them and/or (2) a less stringent assessment of early treatment response in T-LBL than in T-ALL.Given the poor salvage rate of refractory or relapsed T-ALL and T-LBL, optimization of the frontline therapy is critical, and the current study provides a new suggestion for further treatment modifications.However, larger studies in contemporary intensified treatment protocols are required.K E Y W O R D S acute pancreatitis, allergy, asparaginase, children, T-cell acute lymphoblastic leukemia, T-cell lymphoblastic lymphoma

T A B L E 2
Patient characteristics.

Table 2 .
Sex distribution was similar; however, the age at diagnosis was lower in patients with T-ALL (median 8 years for T-ALL vs. 10 years for T-LBL, p < 0.01).The rate of induction failure was similar (7.3% for T-ALL vs. 6.8% for T-LBL, p = 1.00).
The reasons for the discontinuation of L-asparaginase.
Abbreviations: ALL, acute lymphoblastic leukemia; LBL, lymphoblastic lymphoma.T A B L E 3 aOne patient discontinued L-Asparaginase due to both acute pancreatitis and thrombotic event.